Addendum / Correction

The values for LH have been adjusted from the submitted internal report to reflect more

accurately the effects of atrazine on the surge. Although two different dilutions for the 1600

and 1800 hour sampling times had been included in the assay in order to insure that one of the

two diluted samples fell within the linear portion of the standard curve, a number of serum LH

values for the more concentrated dilution fell at the upper, more inaccurate region of the

curve. These were inadvertently included in the analyses. The results of the reanalyzed data

and corrected figures are now presented in the following material, along with small

modifications to the Discussion based upon these LH data.

Results for LH

For LH, 4 days of dosing caused a marginally non

‐ statistically significant effect (p=0.06) on peak

height and a significant suppression In both the AUC and the 2 hour time sampling prior to the

peak (p< 0.05) (Fig. 8). In response to two daily treatments, neither peak height nor AUC were

affected. The single administration of atrazine, on the other hand, caused a marked and

statistically significant elevation in both the peak height of the surge and AUC (p<0.05), with the

100 mg/kg dose resulting in an elevation of 181% over the control AUC.-4 -2 Peak +2 +4 0 1 2 3 4 5 6 7 8 9 10 Ovariectomized / E2 prime - Atrazine 4 Days

Time Relative to Peak 0

100 (10)

(10) mg/kg

0 5 10 15 20 25 30

Atrazine (mg/kg) 0 100 * 35 40Serum LH (ng/ml)Area Under the CurveSerum LH (ng/ml)

* 4 Days Dosing Atrz (1300h)

sampling 1400-1600-1800-2000h

OVX / E2-implant

p = 0.06

54%

-4 -2 Peak +2 +4 +6 0 1 2 3 4 5 6 7 8 9 10 Ovariectomized / E2 prime - Atrazine 2 DaysSerum LH (ng/ml) 0

100 (9)

(9) mg/kg

Time Relative to Peak 0 5 10 15 20 25Serum LH (ng/ml)Area Under the Curve 2 Days Dosing Atrz (1300h)

sampling 1400-1600-1800-2000h

OVX / E2-implant

Atrazine (mg/kg)

0 100 30 35 40

77%

-6 -4 -2 Peak +2 +4 0 1 2 3 4 5 6 7 8 9 10 Ovariectomized / E2 prime - Atrazine 1 Day

Time Relative to Peak 0

100 (11)

(10) mg/kg *

*

0 10 20 30 40

Atrazine (mg/kg) 0 100 * 2 Days Dosing Atrz (1300h)

sampling 1400-1600-1800-2000h

OVX / E2-implantSerum LH (ng/ml)Area Under the CurveSerum LH (ng/ml)

181%

Figure 8. The LH surge (ng/ml + SEM) and areas under the curve in response to 4, 2 and 1 day

of atrazine treatment. The numbers in parentheses indicate the group sizes for each of the

treatments. The numbers within the 100 mg/kg columns at the right indicate the percent

change from the 0 mg/kg group. * p < 0.05.Discussion

The disparity in the influence of a single vs. multiple daily doses of atrazine on the LH surge is

likely attributable to an influence of this herbicide on adrenal progesterone and perhaps

corticosterone secretion. This conclusion is based on what is known about the nature of the

temporal relationships between these two adrenal steroids and the surge. From the data

reported by Fraites et al. (2009), atrazine was able to induce a rapid elevation in progesterone

to approximately 19 ng/ml just 15 minutes after a single 75 mg/kg exposure (greater than 4

‐

fold over controls) (Fig. 9). Although in the present work, we did not observe a significant

difference from controls at 1 hour following the 2 nd or 4 th daily treatments, it is likely that

progesterone was elevated prior to this time. At 15 minutes after the last of 4 daily atrazine

doses, Fraites found that progesterone, while statistically significant at 7 ng/ml, was down to 3

‐

fold over controls, providing evidence of a decline in the stimulation of the adrenal

progesterone response with multiple exposures. It would appear then that in the present

experiment, the lack of a statistically significant atrazine

‐ induced increase may well be due to a

reduction in the magnitude of the rapid increase in progesterone, so that by 1 hour circulating

levels have been sufficiently cleared to approach control concentrations.

Because progesterone and corticosterone are co

‐ secreted from the adrenals, both steroids may

have contributed to a combined effect on the LH surge. However, it appears unlikely that an

increase in blood concentrations of corticosterone was involved in the 1 day effect on the

surge, since previous data obtained employing this OVX / estradiol

‐ primed animal model

showed no difference in the surge when a single injection of corticosterone was given 5 hours

prior to the anticipated LH peak (Fig. 10). In contrast, corticosterone administration on the

previous day produced a statistically significant attenuation in LH, indicating that in the present

experiments a combination of both adrenal steroids, along with a possible non

‐ steroid

‐ linked

mode of action, could have induced the observed response to multiple atrazine dosages.From what is known about the relationships between estradiol and progesterone on the

temporal course of facilitation and inhibition of the LH surge, it doesn’t seem that the present

effects of atrazine are attributable to changes in circulating estradiol. Although Sanderson et al.

(2000) found that estradiol concentrations were elevated following in vitro exposures of the

H295R adrenocortical tumor cells to atrazine, this effect was due to an augmentation in

synthesis through an increase in aromatase activity. In the present experiment, estradiol is

exogenously provided via implant, and the concentration should remain consistent within a

defined range.

In summary, the principal findings of this preliminary report demonstrate two important factors

concerning the HPA/HPG interactions. First, a single dose of atrazine does affect the adrenal

axis by increasing the amount of LH secreted during the afternoon LH surge. The fact that this

same single dose of atrazine increases adrenal hormone secretion (primarily progesterone)

would explain the enhancement of the surge. Second, multiple (four daily) doses of atrazine

resulted in a suppression of the AUC, although peak height was marginally non

‐ statistically

significant. Again, we attribute this suppression in the AUC surge to the activation of adrenal

hormone secretion following each dose of atrazine. As discussed above, the effect of continued

exposure to both progesterone and corticosterone on LH secretion in general and on the LH

surge in particular is well known. Although this preliminary study employed ovariectomized

animals and a single dose of atrazine, it demonstrates that the changes in LH secretion can be

predicted by a recognition of the capacity of this herbicide to affect adrenal steroid hormone

production and the temporal impact of these steroids on LH. These data, when considered in

combination with those results describing the hypothalamic response to atrazine exposure,

depict a complex relationship between the HPG and HPA axes in the initiation of a cascade of

neuroendocrine changes that ultimately lead to altered ovarian function.